Pathogen and host cell interactions play critical roles in the pathogenesis of viral diseases such as AIDS. For a typical viral infection, viruses have to attach to the host cells through cell surface receptors, fuse with host cell membrane, translocate across the cell membrane, uncoat viral particles, synthesize and assemble viral proteins using host protein synthesis machinery, and release from host cells through host exporting machinery. The interplay between the viruses and host cells determine the outcome of viral pathogenesis, ranging from the elimination of viruses to a parasitic or lethal infection. For example, HIV employs a variety of strategies to productively infect human cells. A retrovirus, its life cycle begins by attaching to host cells—the primary target is the CD4+ T helper cells and gaining entry via specific receptors. In the cell, the RNA genome is “reverse” transcribed to its complementary DNA, and then shuttled to the nucleus for its integration in the host genome. This integrated “provirus” then directs the production of new viral RNA and proteins, which self-assemble and then “bud” from the cell as mature—and infections—viral particles, enveloped in plasma membrane. Like all viruses, the HIV is a parasite, unable to catalyze the membrane fission event that drives the budding process. Instead, the nascent virus recruits the cell's membrane sorting machinery to complete this final stage of infection. Such an host and virus interplay has been well demonstrated in individuals, who carry a defective cell surface receptor (CCR5), are completely resistant to HIV infection, elucidating the important roles of host genes and genetic pathways in viral pathogenesis.
Tumor Susceptibility Gene 101 (TSG101, Li et al., 1996, Cell, 85, 319-29) plays important roles in cell growth (Zhong et al., 1998, Cancer Res. 58, 2699-702, Oh et al., 2002, Proc Natl Acad Sci U.S.A 99, 5430-5; Krempler et al., 2002, J. Biol Chem 277, 43216-23; Wagner et al., 2003, Mol. Cell. Biol 23, 150-62; LI et al., 1996, Cell 85, 319-29), cellular protein trafficking (Babst et al., 2000, Traffic 1, 248-58; Bishop et al., 2002, J. Cell Biol. 157, 91-101), and degradation of p53 (Li et al., 2001, proc Natl Acad Sci U.S.A 98, 1619-24; Ruland et al., 2001, Proc Natl Acad Sci U.S.A 98, 1859-64; Moyret-Lalle et al., 2001, Cancer Res 61, 486-8). TSG101 is also widely recognized as a key player in this final stage, inhibition of cellular TSG101 blocks the budding process of HIV. Acting in concert with other cellular factors, TSG101 thus plays an essential role in the budding or spread of HIV viruses. The HIV Gag protein, previously shown to orchestrate viral assembly and budding, binds with high affinity to TSG101, and this Gag/TSG101 interaction is essential for efficient HIV viral assembling and budding, as disruption of the Gag/TSG101 interaction prevents HIV viral budding, and significantly limit the spread of HIV virus.
The final step in the assembly of an enveloped virus assembly requires separation of budding particles from the cellular membranes. Three distinct function domains in Gag, i.e., PTAP in HIV-1 SEQ ID NO.: 7) (Gottlinger et al., 1991,. Proc Natl. Acad. Sci. U.S. A 88, 3195-9; Huang et al., 1995, J. Virol 69, 610-8); PPPY in RSV (SED IS NO.: 8) (Parent et al. 1995, J. Virol 69, 5455-60), MuLV (Yuan et al., 1999, Embo J 18, 4700-10), and M-PMV (Yasuda et al., 1998, J. Virol 72, 4095-103); and YXXL in EIAV SEQ ID NO.: 6) (Puffer et al., 1997, J. Virol 71, 6541-6), have been identified in different retroviruses that are required for this function and have been termed late, or L domains (Wills et al., 1991, Aids 5, 639-54). In HIV-1, the L domain contains a PTAP motif and is required for efficient HIV-1 release (see, e.g., Wills et al., 1994, J. Virol. 68, 6605-6618; Gottlinger et al., 1991, Proc. Natl. Acad. Sci. USA 88, 3195-3199, Huang et al., 1995, J. Virol. 69, 6810-6818). The L domain of HIV-1 p6, especially the PTAP motif, binds to the cellular protein TSG101 and recruits it to the site of virus assembly to promote virus budding (VerPlant et al., 2001, Proc. Natl. Acad. Sci. USA 98: 7724-7729; Garrus et al., 2001, Cell 107:55-65; Martin-Serrano et. Al., 2001, Nature Medicine 7:1313-19; Pornillos et al., 2002, EMBO J. 21:2397-2406; Demirov et al., 2002, Proc Natl. Acad. Sci. USA 99:955-960; PCT Patent Publication WO 02/072790; U.S. Patent Application Publication No. U.S. 2002/0177207). The UEV domain of TSG101 binds the PTAP motif and mono-ubiquitin (Pronillos et al., 2002, Embo J21, 2397-406; Pornillos et al., 2002, Nat. Struct Biol. 9, 812-7), which has also been implicated in HIV-1 budding (Patnaik et al., 2000, Proc. Natl. Acad. Sci. U.S. A 97, 13069-74; Schubert et al., 2000, Proc Natl. Acad. Sci. U.S. A 97, 13057-62; Strack et al., 2000, Proc Natl. Acad. Sci. U.S. A 97, 13063-8). Depletion of cellular TSG101 (Garrus et al., 20001, Cell 107:55-65) or over-expression of a truncated form of TSG101 inhibits HIV-1 release (Demirov et al., 20002, Proc. Natl. Acad. Sci. USA 99-955-960). Under certain circumstances, TSG101 can even substitute for the HIV-1 L domain topromote virus release (Martin-Serrano et al., 2001, Nature Medicine 7:1313-19).
In yeast, the tag 101 ortholog Vps23 has been shown to interact with Vps28 and Vps37 and to form a protein complex named ESCRT-1, which is critical for endosomal protein sorting into the multivesicular body pathway (Katzmann et al., 2001, Cell 106, 145-55). It is hypothesized that this intracellular multivesicular body formation resembles HIV-1 release at the plasma membrane (Garrus et al., 2001, Cell 107:55-65; Patnaik et al., 2000, Proc. Natl. Acad. Sci. U.S.A 97, 13069-74). In mammalian cells, TSG101 interacts with Vps28 to form an ESCRT-I-like complex (Babst et al., 2000, Traffic 1, 248-58; Bishop et al., 2002, J. Cell Biol. 157, 91-101; Bishop et al., 2001, J. Biol. Chem. 276, 11735-42), although the mammalian homolog of Vps37 has not been identified.
Recent studies (Blower et al., 2003, AIDS Rev. 5:113-25; Vadiserri et al., 2003, Nat. Med. 9:881-6) have estimated that s many as 42 million people worldwide have been infected with HIV. The disease has killed more than 3 million people. While the advent of highly potent and targeted combination therapies has slowed the progression of AIDS in industrialized nations, the AIDS pandemic is causing a “human development catastrophe” in developing nations, particularly in Africa, where more than 21 million Africans have been infected. In South Africa alone, the death toll is projected to rise to 10 million by 2015. Related statistics portend a similar crisis in the Asia Pacific region, which, according to United nations' estimates, has more than 7 million HIV-infected individuals. Repercussions from the AIDS pandemic extend well beyond the clinic, which lack the resources to treat the swelling number of recently infected patients (nearly 20% of the adult population in South Africa is infected). Treatment of HIV-infected and gravely ill AIDS patients is stressing the already over-burdened health care systems of Africa and other developing nations. Rose yet, current treatments of HIV—despite their initial success in reducing viral load are beginning to lose their efficacy, as drug-resistant HIV strains are increasingly isolated in newly infected individuals. Further compounding the therapeutic management of HIV disease is the toxicity of current antiretroviral regimens, the magnitude of which complicates the physician's decision to begin and to maintain treatment. Identifying new therapeutic paradigms for the treatment of HIV disease, especially those with mechanisms of action that promise to slow the development of resistance, is indeed a global challenge for the biopharmaceutical industry.
Many viruses are also highly mutable. Methods and compositions relying on targeting such viruses directly are normally not sufficient in the treatment of infections by such viruses. For example, HIV-1 is a highly mutable virus that during the course of HIV-1 infection, the antibodies generated in an infected individual do not provide permanent protective effect due in part to the rapid emergence of neutralization escape variants (Thali et al., 1992, J. Acquired Immune Deficiency Syndromes 5:5911-599). Current therapies for the treatment of HIV-infected individuals focus primarily on viral enzymes involved in two distinct stages of HIV infection, the replication of the viral genome and the maturation of viral proteins. Since the virus frequently mutates, strains resistant to an antiviral inhibitor develop quickly, despite the drug's initial therapeutic effects. In one recent study, the percentage of individuals newly infected with drug-resistant HIV strains increased six fold over a five year period (Little et al., 2002, N. Engl. J. Med. 347:385-94). Further combination therapy, the current standard of care that attacks HIV with inhibitors of both reverse transcriptase and protease, is leading to the development of multi-drug resistant HIV strains. Antiretroviral drugs directed against new HIV-based targets, while of considerable value, do not address this increasingly critical issue. For example, HIV strains resistant to Fuzeon® (enfuvirtide), the newest addition to the anti-HIV armamentarium, have already been isolated from patients. Thus, despite its antiviral potency and novel mechanism of action, drug-resistance is likely to undermine the therapeutic potential of viral fusion inhibitors, like Fuzeon®. There is therefore a need for developing novel therapeutics and preventative measure to combat viral infections such as HIV infection.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.